U.S. patent application number 11/913762 was filed with the patent office on 2008-08-14 for derivatised carbon.
This patent application is currently assigned to ISIS INNOVATION LIMITED. Invention is credited to Richard Guy Compton, Gregory George Wildgoose.
Application Number | 20080190855 11/913762 |
Document ID | / |
Family ID | 34685211 |
Filed Date | 2008-08-14 |
United States Patent
Application |
20080190855 |
Kind Code |
A1 |
Compton; Richard Guy ; et
al. |
August 14, 2008 |
Derivatised Carbon
Abstract
Derivatised carbon is disclosed in which an amino acid or a
derivative thereof is attached to the carbon. Derivatised carbon
may be useful in the detection and removal of metal ions from
liquid media.
Inventors: |
Compton; Richard Guy;
(Oxford, GB) ; Wildgoose; Gregory George; (Oxford,
GB) |
Correspondence
Address: |
KIRTON AND MCCONKIE
60 EAST SOUTH TEMPLE,, SUITE 1800
SALT LAKE CITY
UT
84111
US
|
Assignee: |
ISIS INNOVATION LIMITED
Oxford
GB
|
Family ID: |
34685211 |
Appl. No.: |
11/913762 |
Filed: |
May 5, 2006 |
PCT Filed: |
May 5, 2006 |
PCT NO: |
PCT/GB2006/001643 |
371 Date: |
January 28, 2008 |
Current U.S.
Class: |
210/688 ;
205/789.5; 210/503; 527/207; 528/332; 532/1 |
Current CPC
Class: |
B01J 20/3272 20130101;
B01J 45/00 20130101; C09C 1/56 20130101; C01P 2002/85 20130101;
C01B 32/05 20170801; C02F 2101/103 20130101; C02F 1/283 20130101;
B01J 20/3204 20130101; C02F 2101/20 20130101; B01J 20/3219
20130101; B01J 20/3253 20130101; C09C 1/565 20130101; B01J 20/3251
20130101 |
Class at
Publication: |
210/688 ;
528/332; 527/207; 205/789.5; 532/1; 210/503 |
International
Class: |
C02F 1/28 20060101
C02F001/28; C08G 69/26 20060101 C08G069/26; C01B 31/02 20060101
C01B031/02; C07D 487/00 20060101 C07D487/00; C09C 1/56 20060101
C09C001/56; C02F 1/62 20060101 C02F001/62 |
Foreign Application Data
Date |
Code |
Application Number |
May 6, 2005 |
GB |
0509307.5 |
Claims
1. A derivatised carbon in which an amino acid or a derivative
thereof is attached to the carbon.
2. A derivatised carbon according to claim 1, wherein the amino
acid or derivative thereof is attached to carboxyl groups on said
carbon.
3. A derivatised carbon according to claim 1, wherein a phenylamine
group, substituted by said amino acid or derivative thereof, is
attached to said carbon.
4. A derivatised carbon according to claim 1, wherein the amino
acid is a sulfur-containing amino acid.
5. A derivatised carbon according to claim 4, wherein the amino
acid is cysteine, glutathione, tyrosine or a derivative
thereof.
6. A derivatised carbon according to claim 1, wherein the amino
acid derivative is an oligomer or polymer.
7. A derivatised carbon according to claim 6, wherein the amino
acid derivative is poly-S-benzyl-L-cysteine.
8. A derivatised carbon according to claim 1, wherein the carbon is
graphite powder or glassy carbon spherical powder.
9. A derivatised carbon according to claim 1, wherein the carbon is
glassy carbon spherical powder or pyrolytic graphite.
10. A derivatised carbon according to claim 9, wherein the carbon
is glassy carbon spherical powder and the amino acid or derivative
thereof is cysteine, glutathione, tyrosine or cysteamine.
11. A derivatised carbon according to claim 9, wherein the carbon
is pyrolytic graphite and the amino acid or derivative thereof is
polycysteine or polyglutathione.
12. A derivatised carbon according to claim 1, wherein the carbon
is graphite powder or glassy carbon spherical powder and the amino
acid is cysteine or a derivative thereof.
13. A derivatised carbon according to claim 12, wherein the amino
acid is cysteine, cysteine methyl ester or
poly-S-benzyl-L-cysteine.
14. A method of preparing a derivatised carbon in which carbon is
contacted with a nitrobenzenediazonium compound under conditions
such that a nitrophenyl-derivatised carbon is produced.
15. A method according to claim 14, wherein the carbon is contacted
with the nitrobenzenediazonium compound in the presence of
hypophosphorous acid.
16. A method according to claim 14, further comprising reducing the
nitrophenyl-derivatised carbon to form an aniline-derivatised
carbon.
17. A method according to claim 16, further comprising reacting the
aniline-derivatised carbon with a species to produce a substituted
aniline-derivatised carbon.
18. A method according to claim 17, wherein the aniline-derivatised
carbon is reacted with amino acid or derivative thereof.
19. A method according to claim 18, wherein the amino acid is a
sulfur-containing amino acid and the carbon is graphite powder,
glassy carbon spherical powder, or pyrolytic graphite.
20. A method of preparing a derivatised carbon in which the carbon
is attached directly to the amino acid or derivative thereof via
carboxyl groups on the surface of the carbon, the method comprising
converting carboxyl groups on the surface of the carbon to acyl
halide groups and then contacting the resulting product with the
amino acid or derivative thereof.
21. A method according to claim 20, wherein the acyl halide is acyl
chloride.
22. A method according to claim 20, wherein the amino acid is a
sulfur-containing amino acid and the carbon is graphite powder,
glassy carbon spherical powder, or pyrolytic graphite.
23. A derivatised carbon according to claim 1, wherein the
derivatised carbon is included in a carbon electrode.
24. A derivatised carbon according to claim 23, wherein the carbon
electrode is included in an electrochemical device.
25. A method of removing metal ions from a liquid medium comprising
contacting the medium with derivatised carbon according to claim
1.
26. A method according to claim 25, wherein the metal ions are
selected from Cd(II), Pb(II), Zn(II), Cu (II) and As(III) ions.
27. A method of detecting the presence of metal ions in a liquid
medium comprising subjecting the medium to voltammetric analysis
using an electrochemical device according to claim 24.
28. A method according to claim 25, wherein the medium is an
aqueous medium.
Description
FIELD OF THE INVENTION
[0001] This invention relates to derivatised carbon, in particular
to graphite and other forms of carbon having surfaces chemically
modified to impart desired properties.
BACKGROUND TO THE INVENTION
[0002] The accumulation and release of toxic substances into the
environment, particularly toxic heavy metals, has increased
significantly over the past few decades. The environmental impact
of mining operations and heavy industry has led to the accumulation
of high concentrations of toxic heavy metal ions such as Cu.sup.II,
Cd.sup.II, Pb.sup.II and Hg.sup.II in lakes and rivers, these
pollutants being largely nondegradable and recirculating in nature.
The presence of heavy metals in aquatic media and drinking water
are potentially dangerous to the health of both humans and aquatic
life depending on the exposure levels and chemical form of the
heavy metal. An example of the tragic human consequences of heavy
metal pollution is the widespread poisoning of millions of people
in countries such as Argentina, China, Mexico, Taiwan, India and in
particular Bangladesh, where up to 60% of the Bangladeshi
groundwater contains naturally occurring arsenic concentrations
greatly in excess of the World Health Organisation's (WHO)
guidelines of 10 ppb. As many salts of these heavy metal ions are
water soluble, common physical methods of separation are rendered
ineffective. There is a pressing need to develop a facile, rapid
and inexpensive method of removing toxic heavy metal ions from
aqueous media for use in drinking water filtration and/or
environmental clean up.
[0003] Polypeptides such as poly-L-histidine, poly-L-aspartic acid,
poly-L-glutamic acid and in particular poly-L-cysteine are known to
chelate metal ions such as Cd.sup.II, Pb.sup.II, Ni.sup.II and
Cu.sup.II and have been attached to various substrates and used in
the trace analysis of these metals (Malachowski et al, Anal. Chim.
Acta. 2003, 495, 151; Malachowski et al, Anal. Chim. Acta 2004,
517, 187; Malachowski et al, Pure Appl. Chem. 2004, 76, 777;
Johnson et al, Anal. Chem. 2005, 77, 30; Howard et al, J. Anal. At.
Spectrom. 1999, 14, 1209; and Jurbergs et al, Anal. Chem. 1997, 69,
1893). Biohomopolymers and other peptides possess significant
advantages for metal extraction or reclamation over traditional
techniques such as simple filtration or precipitation, as the
latter are often unable to reduce the concentration of the target
metals to meet strict environmental agency regulations.
[0004] Graphite surfaces can be chemically modified using a variety
of relatively facile techniques such as physisorption and
chemically or electrochemically initiated chemisorption of a given
chemical or biological moiety. Graphite having derivatised surfaces
may be used in a variety of applications, for instance as electrode
materials in battery technology and as sensors. Although reactive
groups such as hydroxyl and carboxyl moieties are known to be
present on the surface of graphitic materials, the use of
chemically derivatised graphite as a solid-state support for
synthetic chemistry applications has been limited.
SUMMARY OF THE INVENTION
[0005] The present invention provides carbon-based solid-state
supports upon which to conduct synthetic, step-wise syntheses. This
allows the derivatisation of the surface of such materials in a
"building-block" fashion, to impart desired properties such as
sensitivity to a target analyte. In this way, species such as amino
acids, peptides, small proteins and nucleic acids can coupled to
carbon (e.g. graphite) particles in a relatively facile manner. By
varying the chemistry of the species that initially derivatises the
carbon surface, various methods of coupling building-block
molecules to the carbon surface are possible. In particular, the
present invention provides derivatised carbon, especially graphite,
to which is attached an amino acid or a derivative thereof. The
amino acid may be monomer (e.g. cysteine) or a polypeptide (e.g.
poly-L-cysteine), which is capable of binding metal ions. The
invention is therefore particularly relevant to the detection and
removal of toxic heavy metals from water and other liquid
media.
[0006] According to a first aspect of the present invention, there
is provided derivatised carbon in which an amino acid or a
derivative thereof is attached to the carbon. The attachment may be
direct or indirect, for example via a phenylamine group.
[0007] The present invention also provides a method of preparing a
derivatised carbon in which the carbon is contacted with a
nitrobenzenediazonium compound under conditions such that a
nitrophenyl-derivatised carbon is produced.
[0008] The present invention also provides a method of preparing
derivatised carbon in which the carbon is attached directly to the
amino acid or derivative thereof via carboxyl groups on the surface
of the carbon, the method comprising converting carboxyl groups on
the surface of the carbon to acyl halide groups and then contacting
the resultant product with the amino acid or derivative
thereof.
[0009] The present invention also provides a carbon electrode
comprising derivatised carbon of the invention.
[0010] The invention further provides an electrochemical device
including an electrode of the invention. The electrochemical device
may be in the form of an electrochemical sensor or reactor.
[0011] In addition, the present invention provides a method of
removing metal ions from a liquid medium comprising contacting the
medium with derivatised carbon of the invention.
[0012] Furthermore, the present invention provides a method of
detecting the presence of metal ions in a liquid medium comprising
subjecting the medium to voltammetric analysis using an
electrochemical device of the invention.
[0013] Derivatised carbon of the invention may be useful in the
detection, removal, sequestration and titration of metal ions from
liquid media, including water and other aqueous media. Such metal
ions include, for instance, Cd(II), Pb(II), Zn(II), Cu(II) and
As(III) ions. The derivatised carbon may be in particulate form,
for example in the form of a powder. Particulate materials such as
graphite powder and glassy carbon powder are desirable because of
their high surface area, which allows them to couple relatively
large amounts of amino acids or derivatives thereof. Derivatised
carbon of the invention may therefore be able to bind a
significantly greater amount of metal ions than known modified
solid-state materials.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows:
[0015] a) consecutive voltammograms showing the response of
4-nitrophenyl-derivatised carbon ("NPcarbon") in pH 6.8 buffer;
[0016] b) overlaid voltammograms of blank graphite powder and
aniline-derivatised carbon ("ANcarbon") in acetonitrile containing
0.1 M tetrabutylammonium perchlorate (TBAP) as supporting
electrolyte; and
[0017] c) consecutive voltammograms showing the response of
4-nitrobenzoic acid-derivatised carbon ("NBANcarbon") in pH 6.8
buffer.
[0018] FIG. 2 shows:
[0019] a) the N.sub.1s region of the X-ray photoelectron
spectroscopy (XPS) spectrum of ANcarbon; and
[0020] b) the N.sub.1s region of the XPS spectrum of
NBANcarbon.
[0021] FIG. 3 shows:
[0022] a) the wide XPS spectrum of
poly-S-benzyl-L-cysteine-derivatised carbon ("PSBCcarbon") and
[0023] b) the wide XPS spectrum of poly-L-cysteine-derivatised
carbon ("PCcarbon").
[0024] FIG. 4 shows linear sweep stripping voltammograms for
Cd.sup.2+ detection with standard additions of Cd.sup.2+. The inset
shows the corresponding standard addition plot.
[0025] FIG. 5 shows the cadmium concentration profile remaining in
a 10 cm.sup.3 sample of river water (original Cd(II) concentration
ca. 1.5 mM) after exposure to 10 mg cysteine
methylester-derivatised glassy carbon ("CysMeO-GC").
[0026] FIG. 6 shows the cadmium concentration profile remaining in
a 10 cm.sup.3 sample of mineral water (original Cd(II)
concentration 50 ppb) after exposure to 10 mg CysMeO-GC.
[0027] FIG. 7 shows the copper concentration profile remaining in a
10 cm.sup.3 sample of river water after exposure to 10 mg CysMeO-GC
for varying times.
[0028] FIG. 8 shows the concentration of As(III) remaining after
exposure to 10 mg of PCcarbon powder, stirred for specified lengths
of time. The curve shows a first order exponential decay fitted to
the data.
[0029] FIG. 9 shows the concentration of As(III) remaining after
exposure to 10 mg of CysMeO-GC powder, stirred for specified
lengths of time. The curve shows a first order exponential decay
fitted to the data.
[0030] FIG. 10 shows the concentration of As(III) remaining after
exposure to 200 mg of CysMeO-GC powder to a 200 ppb As(II)
solution, stirred for specified lengths of time. The curve shows a
first order exponential decay fitted to the data.
[0031] FIG. 11 shows the concentration of As(III) remaining after
exposure to 200 mg of CysMeO-GC powder to a 120 ppb As(III)
solution in a Bangladeshi water sample, stirred for specified
lengths of time. The curve shows a first order exponential decay
fitted to the data.
[0032] FIG. 12 shows anodic stripping voltammograms of a 120 ppb
As(III) Bangladeshi water sample exposed to 200 mg of CysMeO-GC
spherical powder and stirred for 30 minutes. Linear sweep
voltammetry (LSV) was performed at 100 mV/s, and standard additions
of 2.4.times.10.sup.-7 M were used.
[0033] FIG. 13 shows an XPS spectrum of L-cysteine methyl
ester-modified carbon powder ("CysOMe-carbon").
[0034] FIG. 14 shows an baseline-corrected XPS spectrum of
CysOMe-carbon powder after exposure to As.sup.III showing the
region of interest from 120 to 260 eV. The dotted lines show the
Gaussian peak fitting performed using the MicroCal Origin software
package.
[0035] FIG. 15 shows overlaid concentration-time profiles for the
removal of Cd.sup.II from a ca. 55 .mu.M solution of
Cd(NO.sub.3).sub.2 in pH 5.0 acetate buffer comparing the efficacy
of CysOMe-GC and CysOMe-carbon powder adsorbents.
[0036] FIG. 16 shows a concentration-time profile for the removal
of trace amounts of As.sup.III to below the WHO recommended limit
of 10 ppb.
[0037] FIG. 17 shows overlaid Cd.sup.II linear sweep anodic
stripping voltammetry (LSASV) voltammograms with increasing 1 .mu.M
standard additions of Cd.sup.II (0-20 .mu.M). The inset shows the
corresponding standard addition plot.
[0038] FIG. 18 shows overlaid As.sup.III LSASV voltammograms with
increasing 0.22 .mu.M standard additions of As.sup.III (0 to 2.2
.mu.M). The inset shows the corresponding standard addition
plot.
DESCRIPTION OF VARIOUS EMBODIMENTS
[0039] The invention provides derivatised carbon to which is
attached an amino acid or a derivative thereof. The amino acid or
derivative may be attached directly or indirectly (i.e. via a
linker) to the carbon. Of particular mention is carbon to which the
amino acid or derivative is attached via a carboxyl or phenylamine
group present on the carbon.
[0040] In one embodiment, the amino acid is a sulfur-containing
amino acid, for instance, cysteine, glutathione, tyrosine or a
derivative thereof. The sulphur-containing amino acid may have
pendant thiol or thiol-like groups. The amino acid may be in the
form of an ester, e.g. a methyl or ethyl ester, a particular
example being L-cysteine methyl ester. Derivatives of amino acids
include oligomers and polymers of amino acids. By way of example, a
cysteine derivative may be polycysteine or cysteamine, while a
glutathione derivative may be polyglutathione. An exemplary
polymeric amino acid is an S-benzyl protected homopolymer
containing 50 to 100 cysteine residues per polymer chain. The amino
acid, or derivative thereof, may be protected or unprotected, an
example being a polycysteine such as poly-S-benzyl-L-cysteine.
[0041] The carbon may be in particulate form, for example in the
form of a powder. A particulate carbon may comprise particles
having a diameter of between 1 and 100 .mu.m, e.g. between 2 and 50
.mu.m. Of particular mention are graphite powder, glassy carbon
spherical powder and pyrolytic graphite forms. Alternatively, the
carbon may be in the form of carbon nanotubes, for instance,
multiwalled carbon nanotubes (MWCNTs).
[0042] Examples of derivatised carbons of the invention include
glassy carbon modified with cysteine, glutathione or cysteamine or
a derivative thereof, and a carbon powder modified with
polycysteine or polyglutathione. It will be appreciated that the
invention extends to other amino acid polymers and derivatives and
also to monomers of amino acids and their thiol-containing
derivatives, such as cysteine, coupled to glassy carbon. Particular
examples include carbon powder (e.g. graphite powder or glassy
carbon spherical powder) derivatised with cysteine or a derivative
thereof (e.g. an ester of cysteine such as cysteine methyl ester,
or a polymer of cysteine such as polycysteine or
poly-S-benzyl-L-cysteine).
[0043] The derivatised carbon may be obtained by contacting carbon
with a nitrobenzenediazonium compound under conditions such that a
nitrophenyl-derivatised carbon is produced. The reaction may be
carried out in the presence of a suitable reagent such as
hypophosphorous acid. The nitrophenyl-derivatised carbon may be
reduced to form an aniline-derivatised carbon. The product may be
further reacted to produce a substituted aniline-derivatised
carbon. In particular, the aniline-derivatised carbon may be
reacted with an amino acid or derivative thereof (e.g. a
polycysteine such as poly-S-benzyl-L-cysteine).
[0044] Derivatised carbon may also be obtained by converting
carboxyl groups present on the surface of a carbon to acyl halide
groups and then contacting the resulting product with an amino acid
or derivative thereof. The acyl halide may be, for example, acyl
chloride. Any carboxyl groups present on the amino acid or
derivative thereof may be protected.
[0045] Derivatised carbon of the invention may be used in the
detection (e.g. the electrochemical detection), titration or
removal of metal ions from liquid media. The metal ions may be, for
instance, one or more of Cd(II), Pb(II), Zn(II), Cu(II) and As(III)
ions. The liquid medium may be, for instance, an aqueous
medium.
[0046] Derivatised carbon of the invention, especially cysteine- or
polycysteine-derivatised carbon, may be useful in the detection of
arsenic. For example, the carbon may be provided in a relatively
expensive drinking water filtration device. Conversely, to the
extent that a derivatised carbon of the invention is selective for
metal ions other than As(III), it may be incorporated into an
arsenic sensor in order to remove ions such as Cu(II), which
interfere in As(III) detection. Accordingly, the invention may
provide inexpensive and attractive materials for use in water
clean-up, the recovery or extraction of metals from effluents, and
drinking water filtration, where natural supplies are often
contaminated by toxic heavy metals such as arsenic and cadmium.
[0047] The invention further provides materials which may be useful
in metal sequestration. By way of example, polycysteine anchored on
carbon typically has a much higher metal uptake (per gram of
material) than known substrates such as glass, polymer beads and
the like. The density of sequestration units per surface area may
also be much greater than for prior art substrates where nano-scale
modification is used (e.g. in the case of nanotubes) is used, due
to an increase in active surface area. Hence both the
thermodynamics and the kinetic rate of metal ion uptake may be
enhanced.
[0048] In particular, the present invention provides a solid-state
support material in which the support is provided by coupling a
biohomopolymer, in particular a polypeptide selected from
poly-L-histidine, poly-L-aspartic acid, poly-L-glutamic acid and
especially poly-L-cysteine, to graphite powder. As mentioned above,
such polymers are known to chelate toxic heavy metals such as
cadmium, lead, nickel and copper with very little affinity for
alkali and alkaline earth metals such as sodium and calcium. A
cysteine-, poly-L-cysteine-derivatised graphite powder of the
invention may be used to quantitatively titrate metal ions, such as
Cd(II) ions, from aqueous media. Due to the high surface area of
graphite powder and the ability to couple large amounts of amino
acid to it, cysteine- or polycysteine-modified carbon may chelate
far greater amounts of Cd(II) ions than poly-L-cysteine attached to
any other solid-state support material. Thus, derivatised carbon of
the invention is particularly suited for use in toxic heavy metal
recovery from industrial effluents, environmental cleanup and
drinking water filtration.
[0049] The following Examples illustrate the invention.
EXAMPLE 1
Derivatisation of Graphite Powder with Poly-L-Cysteine
Reagents and Chemicals
[0050] With the exception of potassium chloride (purchased from
Riedel de Haen), all reagents were obtained from Aldrich and were
of the highest grade available and used without further
purification. The synthetic graphite powder used consisted of
irregularly shaped particles of between 2 and 20 .mu.m diameter and
was purchased from Aldrich. All aqueous solutions were prepared
using deionised water from an Elgastat UHQ grade system (Elga) with
a resistivity of not less than 18.2 M.OMEGA. cm.
[0051] Solutions of known pH in the range pH 1.0 to pH 12.0 were
prepared in deionised water as follows: pH 1.0, 0.10 M HCl; pH 1.7,
0.1 M potassium tetraoxalate; pH 4.6, 0.10 M acetic acid +0.10 M
sodium acetate; pH 5.04, 0.5 M sodium acetate; pH 6.8, 0.025 M
Na.sub.2HPO.sub.4+0.025 M KH.sub.2PO.sub.4; pH 9.2, 0.05 M disodium
tetraborate; pH 10.5, 0.1 M disodium tetraborate; and pH 12.0, 0.01
M sodium hydroxide. These solutions contained in addition 0.10 M
KCl as supporting electrolyte. pH measurements were performed using
a Hanna pH213 pH meter.
Instrumentation
[0052] Electrochemical measurements were recorded using a pautolab
computer controlled potentiostat (Ecochemie) with a standard
three-electrode configuration. Electrochemical experiments were
carried out in a glass cell of volume 25 cm.sup.3. Either a basal
plane pyrolytic graphite electrode (bppg, 5 mm diameter, Le
Carbone) or boron doped diamond electrode (BDD, 3 mm diameter,
Windsor Scientific Ltd.) electrode acted as the working electrode.
A platinum coil (99.99%, Goodfellow) acted as the counter
electrode. The cell assembly was completed using a saturated
calomel electrode (SCE, Radiometer) as the reference electrode
unless otherwise stated. All electrochemical experiments were
carried out after degassing the solution using pure N.sub.2 gas
(BOC gases) for 30 minutes and were conducted at 20.+-.2.degree.
C.
[0053] X-ray photoelectron spectroscopy (XPS) of the
4-nitrophenyl-derivatised carbon after reduction with Sn/HCl and
after the coupling of 4-nitrobenzoic acid was performed on a
Scienta ESCA300 instrument using X-ray radiation from the aluminium
Ka band (hv=1486.7 eV), source setting 14 hv, 200 mA. All spectra
were recorded using a pass energy of 150 eV and a take off angle of
90.degree.. A slit width of 1.9 mm was used, unless otherwise
stated. The base pressure in the analysis chamber was maintained at
not more than 2.0.times.10.sup.-9 mbar.
[0054] XPS of the S-benzyl-protected poly-L-cysteine and the
deprotected poly-L-cysteine was performed on a VG Clam 4 MCD
analyzer system, using X-ray radiation from the Al K.sub..alpha.
band (hv=1486.7 eV). All XPS experiments were recorded using an
analyzer energy of 100 eV with a take-off angle of 90.degree.. The
base pressure in the analysis chamber was maintained at not more
than 2.0.times.10.sup.-9 mbar. Each derivatised carbon sample
studied was mounted on a stub using double sided adhesive tape and
then placed in the ultra-high vacuum analysis chamber of the
spectrometer. To prevent the sample from becoming positively
charged when irradiated due to emission of photoelectrons, the
sample surface was bombarded with an electron beam (10 eV) from a
"flood gun" within the spectrometer's analysis chamber. Analysis of
the resulting spectra was performed using Microcal Origin 6.0.
Assignment of spectral peaks was determined using the UKSAF and
NIST databases.
General Reaction Scheme
[0055] Scheme I illustrates synthetic routes for derivatising
graphite powder showing the principle behind the "building-block"
chemistry and the coupling of poly-L-cysteine to graphite
powder:
##STR00001##
Derivatisation of Graphite Powder with 4-nitrophenyl to Form
NPcarbon
[0056] First 0.5 g of graphite powder was stirred into 10 cm.sup.3
of a 5 mM solution of Fast Red GG (4-nitrobenzenediazonium
tetrafluoroborate), to which 50 cm.sup.3 of hypophosphorous acid
(H.sub.3PO.sub.2, 50% wlw in water) was added. Next, the solution
was allowed to stand at 5.degree. C. for 30 minutes with gentle
stirring, after which the solution was filtered by water suction
and washed with deionised water to remove any excess acid and
finally with acetonitrile to remove any unreacted diazonium salt.
The 4-nitrophenyl-derivatised graphite powder ("NPcarbon") was then
air-dried by placing inside a fume hood for a period of 12 hours
after which they were stored in an airtight container prior to use
(Pandurangappa et al, Analyst, 2002, 127 1568; and Pandurangappa et
al, Analyst, 2003, 128, 473).
Reduction of NPcarbon to Form ANcarbon
[0057] NPcarbon powder (1.02 g) and tin (1.63 g, 13.7 mmol) were
suspended in water (12 mL). Concentrated hydrochloric acid (4.5 ml,
53.8 mmol) was added and the mixture heated to reflux. The reaction
mixture was stirred at 100.degree. C. under an atmosphere of argon.
After 18 h the mixture was filtered and the solid washed with
hydrochloric acid (100 mL of a 1M aqueous solution), methanol (100
mL), potassium hydroxide (50 mL of a 1M aqueous solution) and
methanol (50 mL). The solid was dried in vacuo to afford a black
powder (180.4 mg) of the reduced form of NPcarbon consisting of
p-aniline moieties covalently derivatised to the graphite surface
("ANcarbon").
Coupling of 4-nitrobenzoic Acid to ANcarbon
[0058] ANcarbon (500 mg), 1-hydroxybenzotriazole hydrate (HOBt, 670
mg, 5.0 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate (PyBop, 2.6 g, 5 mmol) and p-nitrobenzoic acid
(840 mg, 5 mmol) were placed in a flask and DMF (8 mL) added. Ethyl
diisopropylamine (1.7 mL, 10 mmol) was added. The reaction mixture
was stirred under argon at room temperature. After 18 h the mixture
was filtered and the solid washed with methanol (50 mL),
acetonitrile (50 mL) and DCM (50 mL). The solid was dried in vacuo
to afford a black powder consisting of 4-nitrobenzoic acid coupled
to the ANcarbon surface via an amide linkage ("NBANcarbon").
Voltammetric and XPS Characterisation of NPcarbon, ANcarbon and
NBANcarbon
[0059] Voltammetric characterisation of the derivatised NPcarbon,
ANcarbon and NBANcarbon was carried out over the range pH 1.0 to pH
12.0, after first separately abrasively immobilising each
derivatised carbon onto the surface of a bppg electrode as
described in Leventis et al, Talanta, 2004, 63, 1039.
[0060] FIG. 1a shows the voltammetry of NPcarbon at pH 6.8. Upon
first scanning in a reductive direction a large reduction wave was
observed at ca. -0.685 V vs. SCE labelled as "System I" in FIG. 1a.
When the scan direction was reversed at -1.0 V vs. SCE, no
corresponding oxidation wave for System I was observed, indicating
that the process was electrochemically irreversible. However, an
oxidation wave was observed at ca. +0.025 V vs. SCE. On subsequent
scans the corresponding reduction wave is observed at ca. -0.095 V
vs. SCE corresponding to an electrochemically almost-reversible
process, termed "System II". The electrochemically irreversible
System I is not present in subsequent scans indicating that all the
4-nitrophenyl moieties have been reduced.
[0061] The observed voltammetric behaviour and their wave-shapes
are consistent with previous studies of NPcarbon (Pandurangappa et
al, Analyst, 2002, 127, 1568) and corresponds to the
electrochemical reduction of the surface-bound nitro groups in
aqueous media. Scheme 2 illustrates this behaviour for the generic
example of nitrobenzene itself (Pandurangappa et al, Analyst, 2002,
127, 1568; and Rubinstein, J. Electroanal. Chem., 1971, 29,
309):
##STR00002##
[0062] In this mechanism, System I corresponds to the chemically
and electrochemically irreversible reduction of the nitro group in
a four-electron, four-proton process to form the arylhydroxylamine.
This then undergoes an electrochemically almost-reversible
two-electron, two-proton oxidation (System II) to form the
arylnitroso species. This voltammetric behaviour was observed at
every pH studied, although, due to concomitant proton transfer, the
peak potentials for both Systems I and II depended on pH and vary
by 55.4 and 54.4 mV/pH unit respectively in a linear, Nernstian
fashion over the range pH 1.0 to pH 12.0 in agreement with previous
studies.
[0063] A well established voltammetric characterisation protocol
(Leventis et al, Talanta 2004, 63, 1039; and Wildgoose et al,
Talanta, 2003, 60, 887), was then carried out over the
almost-reversible System II at each pH studied and confirmed that
the 4-nitrophenyl moieties were indeed confined to the surface of
the graphite particles.
[0064] Voltammetric characterisation of ANcarbon revealed that no
voltammetric waves corresponding to either System I or II were
observed. Thus it could be concluded that all the 4-nitrophenyl
groups were reduced to the corresponding aniline-like moieties.
Voltammetry of ANcarbon in acetonitrile containing 0.1 M
tetrabutylammonium perchlorate (TBAP) showed, in the first scan, an
oxidative wave at ca. +0.700 V vs. a silver pseudo-reference
electrode at potentials corresponding to the one-electron oxidation
of aniline to its radical cation (FIG. 1b).
[0065] The ANcarbon was further characterised using XPS. FIG. 2a
shows that a single peak is observed in the N.sub.1s region of the
spectrum with a binding energy of 400.1 eV consistent with an
aromatic amine moiety. No signals at binding energies corresponding
to photoelectrons emitted from the N.sub.1s or O.sub.1s levels
within a nitro moiety were observed.
[0066] Voltammetric characterisation of the NBANcarbon revealed
that the expected characteristic reduction of the nitro group is
once again observed and that the voltammetry corresponds to a
surface bound species (FIG. 1c). FIG. 2b shows the N.sub.1s region
of the XPS spectrum of NBANcarbon. Two peaks are observed with
binding energies of 400.6 eV and 405.4 eV and an almost 1:1 ratio
of peak heights. Comparison with XPS databases confirms that these
peaks correspond to nitrogen atoms in the amide and nitro groups
respectively. Furthermore, Gaussian deconvolution of the O.sub.1s
region of the spectrum (not shown) reveals peaks with binding
energies of 530.7 eV and 533.6 eV consistent with oxygen atoms
within an amide and an aromatic nitro group respectively. In light
of these results, it can be concluded that coupling takes place
solely between the 4-nitrobenzoic acid molecules and the
aniline-like moieties on the surface of ANcarbon.
Coupling of Poly-S-benzyl-L-cysteine to ANcarbon to Form
PSBCcarbon
[0067] Poly-S-benzyl-L-cysteine (PSBC, 170 mg, 0.02 mmol) was
dissolved in 1,4-dioxane (3 ml). Trimethylsilyl chloride (5.6
.mu.L, 0.04 mmol) in DMF (3 mL) was added to increase the
solubility of the peptide homopolymer. The reaction mixture was
stirred under argon at 50.degree. C. After 1 h the reaction mixture
was cooled to room temperature. Ethyl diisopropylamine (6.5 .mu.L,
0.04 mmol) was added and the mixture cooled to 0.degree. C. before
addition of 9-fluoroenylmethoxycarboxyl chloride (Fmoc, 5.7 mg,
0.02 mmol). The mixture was allowed to warm to room temperature.
After 1 h 30 min the solvent was removed in vacuo to afford a white
solid. To the residue was added 1-hydroxybenzotriazole hydrate
(HOBt, 4.2 mg, 0.2 mmol),
benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate
(PyBop, 11.6 g, 0.02 mmol), ANcarbon (104 mg) and DMF (10 mL).
Ethyl diisopropylamine (17.7 .mu.L, 0.04 mmol) was added. The
reaction mixture was stirred under argon at room temperature. After
19 h the mixture was filtered and the solid washed with DMF (10
mL), methanol (10 mL), acetonitrile (50 mL) and DCM (50 mL). The
solid was dried in vacuo to afford a black powder (200 mg)
consisting of S-benzyl protected poly-L-cysteine coupled to
ANcarbon via an amide linkage ("PSBCcarbon").
Deprotection of PSBCcarbon
[0068] Deprotection of the thiol groups in the poly-L-cysteine was
achieved using a Birch reduction process. Liquid ammonia (ca. 10
mL) was condensed into a flask containing PSBCcarbon (124 mg) and
sodium (120 mg, 5.2 mmol). The solution was stirred under argon at
-78.degree. C. After 20 min 1-butanol (0.3 mL) was added and the
reaction stirred for a further 5 min before being allowed to warm
to room temperature. Once the ammonia had evaporated ammonium
chloride (ca. 4 mL of a saturated aqueous solution) was added to
quench the reaction. The suspension was filtered and the solid
washed with water (20 mL), methanol (20 mL) and DCM (20 mL). The
solid was dried in vacuo to afford a black powder (106 mg)
consisting of poly-L-cysteine coupled to ANcarbon via an amide
linkage ("PCcarbon").
XPS Characterisation of PSBCcarbon and PCcarbon
[0069] FIGS. 3a and 3b show the resulting XPS spectra for
PSBCcarbon and PCcarbon respectively. Two peaks with binding
energies of 162.5 eV and 226.5 eV corresponding to photoelectrons
emitted from the S.sub.2p3\2 and the S.sub.2s levels were observed
in the PSBCcarbon in excellent agreement with literature values for
S-benzyl protected polycysteine. In the deprotected PCcarbon the
binding energies of the S.sub.2p3/2 and the S.sub.2s photoelectrons
were shifted slightly to 163.5 eV and 227.5 eV, again in excellent
agreement with literature values for the free thiol in
polycysteine. For both the PSBCcarbon and the PCcarbon the O.sub.1s
and N.sub.1s peaks are located at 531.5 eV and 400.5 eV
respectively are dominated by the contribution from the amide
linkages in the polycysteine and are in excellent agreement with
literature values. Elemental analysis of both the PSBCcarbon and
PCcarbon samples revealed that the relative amounts of
poly-L-cysteine coupled to the graphite surface did not change
after deprotection of the thiol groups using a Birch reduction with
the total sulphur oxygen and nitrogen signals accounting for ca.
7.+-.1.4% each of the surface elemental composition, indicating
that a relatively large amount of polycysteine was coupled to the
surface. Thus it can be concluded that poly-L-cysteine coupled to
ANcarbon and remained coupled after carrying out a Birch reduction
to deprotect the thiol groups within the poly-L-cysteine.
EXAMPLE 2
Quantitative Analysis of Cadmium in Aqueous Media Using
PCcarbon
[0070] The uptake of Cd.sup.2+ from aqueous solutions was monitored
electrochemically using a linear-sweep stripping voltammetric (LSV)
stripping protocol at a boron doped diamond (BDD) electrode
developed by Banks et al, Talanta, 2004, 62, 279).
[0071] The optimised pH for Cd.sup.2+ detection is pH 5 and
therefore a 0.05M sodium acetate buffer (pH 5.04) was used for both
the chelation of Cd.sup.2+ by the PCcarbon and the LSV detection of
the amount of Cd.sup.2+ chelated. The LSV protocol for cadmium
detection involved depositing the Cd.sup.2+ on the BDD electrode as
Cd.sup.0 by holding the potential at -1.5 V vs. SCE for 60 s whilst
stirring the solution. LSV was then carried out by scanning the
potential from -1.1 V to -0.3 V at 100 mVs.sup.-1 and a cadmium
stripping peak observed at ca. -0.8 V vs. SCE. To verify the
accuracy of this protocol, a "blind" solution of Cd(NO.sub.3).sub.2
was analysed by standard additions of 5 nM Cd.sup.2+ and a standard
addition plot of peak height vs. Cd.sup.2+ concentration
constructed.
[0072] FIG. 4 shows the overlaid resulting LSV voltammograms for
increasing amounts of Cd.sup.2+ and the resulting standard addition
plot (inset). The Cd.sup.2+ concentration was determined by the LSV
protocol to be 20.5 nM.+-.0.1 nM with a limit of detection
(3.sigma.) of 0.2 nM. The actual Cd.sup.2+ concentration was 20
nM.+-.0.1 nM demonstrating that the LSV protocol was an accurate
method for trace Cd.sup.2+ determination over the concentration
range 1-100 nM.
[0073] In order to measure the amount of Cd.sup.2+ chelated by
PCcarbon a 1 mM Cd(NO.sub.3).sub.2 solution was made up in pH 5
sodium acetate buffer. A 10 .mu.L sample of this was then removed
and diluted by a factor of 10.sup.5 in order for the initial
Cd.sup.2+ concentration to be measured by the LSV protocol. Next 5
mg, 10 mg and 20 mg of PCcarbon were added to 10 cm.sup.3 of the 1
mM, 2 mM and 3 mM Cd(NO.sub.3).sub.2 respectively and stirred for
ten minutes. The PCcarbon was then filtered off and again a 10
.mu.L sample of the filtrate was removed and diluted before the
amount of Cd.sup.2+ remaining in the sample was measured using the
LSV protocol. This procedure was repeated three times for each
amount of PCcarbon added
[0074] Table 1 shows the amount of Cd.sup.2+ chelated for varying
masses of PCcarbon. The experiments were repeated with the length
of time the PCcarbon was stirred with Cd.sup.2+ varied from ten
minutes to 12 hours. Increasing the exposure time of Cd.sup.2+ to
PCcarbon was not found to increase the amount of Cd.sup.2+
chelated. A similar experiment was carried out with blank graphite
powder for comparison. The uptake of Cd.sup.2+ by blank graphite
powder was not measurable. From the results presented in Table 1 it
was possible to calculate that PCcarbon chelates 1218
.mu.mol.+-.200 .mu.mol of Cd.sup.2+ per gram of PCcarbon.
TABLE-US-00001 TABLE 1 [Cd.sup.2+] Mass of Cd.sup.2+ Mass of
Initial [Cd.sup.2+] Final [Cd.sup.2+] chelated by chelated
PCcarbon/ determined determined PCcarbon/ per mg of mg by LSV/mM by
LSV/mM mM PCcarbon/mg 5 1.1 0.5 0.6 0.14 10 2.0 0.6 1.4 0.16 20 3.1
0.5 2.6 0.14
[0075] The amount of Cd.sup.2+ chelated by varying masses of
PCcarbon exposed to 10 cm.sup.3 solutions of varying Cd.sup.2+ for
10 minutes.
[0076] The uptake of Cd.sup.2+ by PCcarbon was shown to be up to
one hundred times greater per gram than previous studies where
polycysteine was coupled to other substrates (Jurbergs et al, Anal.
Chem., 1997, 69, 1893; Malachowski et al, Pure Appl. Chem., 2004,
76, 777; Johnson et al. Anal. Chem. 2005, 77,30; and Howard et al,
J. Anal. At. Spectrom., 1999, 14, 1209). Without wishing to be
bound by theory, it is believed that this may be due to the large
surface area of graphite powder and the large proportion of
4-nitrophenyl groups that can be coupled to the numerous
edge-plane-like defect sites on the carbon surface, allowing a far
greater amount of polycysteine to be coupled to graphite powder
than to other solid-state supports. Furthermore, the quantitative
titration of Cd.sup.2+ ions by PCcarbon occurs rapidly (<10
minutes) upon exposure of the PCcarbon to the cadmium (II)
solution.
[0077] Previous studies have demonstrated that Cd.sup.2+ can be
quantitatively recovered from polycysteine using nitric acid as a
result of tertiary conformational changes, rather than simple
proton exchange with the thiol groups (Howard et al, J. Anal. At.
Spectrom., 1999, 14, 1209; and Miller et al, Anal. Chem., 2001, 73,
4087). Cadmium ions were recovered from the PCcarbon by stirring
the filtered PCcarbon samples in 1M HNO.sub.3. After stirring each
sample of PCcarbon in 10 cm.sup.3 1.0 M HNO.sub.3 for either 30
minutes or 5 hours, the suspension was filtered. A 10 .mu.L sample
of the filtrate was removed, and diluted in pH 5 buffer before the
amount of Cd.sup.2+ remaining in the sample was measured using the
LSV protocol. In each instance, irrespective of whether the sample
was treated for 30 minutes or 5 hours, 40%.+-.10% of the chelated
Cd.sup.2+ was recovered. This is in agreement with the studies of
Howard et al, who found that polycysteine exhibits both weak and
strong binding sites for Cd.sup.2+.
EXAMPLE 3
Derivatisation of Graphite Powder and MWCNTs with Tyrosine
[0078] 4-Nitrophenyl groups were coupled to graphite and MWCNTs via
the diazonium salt chemistry described in Example 1. The nitro
group was reduced with Sn/HCl to produce aniline-modified carbon
and MWCNTs. The aniline group was then diazotised and coupled to
tyrosine to produce a material capable of metal chelation and also
a route for further coupling amino acid- or thiol-containing
molecules to the tyrosine-modified carbon and MWCNTs. The amine
groups of the aniline moieties on the surface of the derivatised
carbon and MWCNTs were also converted to thiol groups, for use in
metal chelation/recovery.
EXAMPLE 4
Derivatisation of Glassy Carbon Powder with L-cysteine Methyl
Ester
[0079] 2 g Glassy carbon spherical powder (GC, 10-20 .mu.m
diameter, Type I, Alfa Aesar) was stirred with 10 cm.sup.3
SOCl.sub.2 for 1 hour after which it was washed with dry
CH.sub.3Cl. This converts the carboxyl surface groups to the
acyl-chloride analogues. This material was then reacted with 0.5 g
of L-cysteine-methylester hydrochloride salt (Sigma-Aldrich) in 10
cm.sup.3 dry CH.sub.2Cl.sub.2, with stirring and the slow addition
of 0.27 cm.sup.3 Et.sub.3N. The reaction mixture was then stirred
for 12 hours (overnight) to produce L-cysteine
methylester-derivatised GC spherical powder ("CysMeO-GC"). This
process is illustrated in Scheme 3:
##STR00003##
[0080] In a similar procedure, glassy carbon spherical powder was
coupled with glutathione (reduced form, <99%, Aldrich) and
cysteamine hydrochloride salt (Acros Organics).
EXAMPLE 5
Removal of Cadmium from Water Using CysMeO-GC Powder
Detection of Cadmium
[0081] The linear sweep voltammetry (LSV) stripping protocol used
was based on a previous detection protocol (Kruusman et al,
Electroanalysis, 2004, 16, 399). A boron doped diamond electrode
(BDD, diameter of 3 mm, Windsor Scientific) was used as the working
electrode, with a platinum coil and saturated calomel electrode
(SCE, Radiometer) acting as counter and reference electrodes
respectively. The electrochemical experiments were carried out
using a computer controlled potentiostat (.mu.Autolab) in pH 5.04
0.05M sodium acetate buffer with 0.1 M KCl added as supporting
electrolyte.
[0082] LSV detection of Cd(II) was carried out using the following
parameters: a 10 .mu.L aliquot of the sample to be tested was added
to 10 cm.sup.3 of the sodium acetate buffer. Cadmium was deposited
onto the BDD electrode at a potential of -1.5 V vs. SCE, for 60 s
with stirring. The potential was then swept at 100 mVs.sup.-1 from
-1.1 V to -0.6 V vs. SCE with a cadmium stripping peak observed at
ca. -0.780 V vs. SCE. Standard additions of 0.1 .mu.M Cd(II) were
then added over the range 0.1-1.0 .mu.M and a corresponding
addition plot was constructed and used to calculate the background
Cd(II) concentration in the original sample.
Removal of Cadmium from River Water
[0083] A sample of river water was taken (untreated) from the River
Cherwell in Oxford. A 10 cm.sup.3 sample of this river water was
spiked to produce a cadmium(II) concentration of ca. 1.5 mM to
simulate an environmentally disastrous spillage of toxic cadmium
waste. This connection is the calculated average Cd(II)
concentration in the River Neva which flows through St Petersburg,
Russia and which is well known to be heavily polluted. 10 mg of
CysMeO-GC powder was then added to this "real" matrix sample and
stirred. The sample was filtered and a 10 .mu.L aliquot removed for
analysis using the LSV Cd(II) stripping protocol given above every
after 5 minutes and then at every 10 minute interval for 1
hour.
[0084] FIG. 5 shows the resulting Cd(II) concentration profile. It
is apparent that ca. 87% of the Cd(II) was removed from the sample
by 10 mg of CysMeO-GC powder. The residual Cd(II) concentration was
approximately half that of the calculated drinking water
concentration of Cd(II) in the St Petersburg water supply out of
the tap, which is still above the WHO, EU and EPA guidelines.
CysMeO-GC powder may be used as a cheap and highly effective
material for use in environmental clean up and/or metal ion
sequestration.
Removal of Cadmium from Mineral Water
[0085] The contamination of drinking water supplies was simulated
by spiking a 10 cm.sup.3 sample of Evian mineral water Cd(II) to
produce a Cd(II) concentration of 50 ppb (parts per billion), which
is ten times the EPA recommended maximum limit for drinking water.
This "real" matrix was then stirred with 10 mg CysMeO-GC powder and
analysed as described above. The resulting removal of Cd(II) is
shown in FIG. 6.
[0086] Within ten minutes of exposure to CysMeO-GC powder the
Cd(II) concentration in the mineral water was below the EPA
recommended maximum limit of 5 ppb. Cys-GC is therefore an
excellent material for use in drinking water filtration to remove
toxic heavy metals such as Cd(II).
EXAMPLE 6
Removal of Copper from Water Using CysMeO-GC Powder
Detection of Copper
[0087] The square wave voltammetry (SWV) stripping protocol used
was based on a previous detection protocol (Banks et al, Phys.
Chem. Chem. Phys., 2003, 5, 1652). A 50 .mu.m diameter gold disc
electrode (<99.99%, Goodfellow) was used as the working
electrode, with a platinum coil and saturated calomel electrode
(SCE, Radiometer) acting as counter and reference electrodes
respectively. The electrochemical experiments were carried out
using a computer controlled potentiostat (.mu.Autolab) in pH 2.00
0.1 M phosphoric acid (H.sub.3PO.sub.4) buffer with 0.1 M KCl added
as supporting electrolyte.
[0088] SWV detection of Cu(II) was carried out using the following
parameters: frequency 50 Hz, step potential 2 mV, amplitude 25 mV.
A 0.5 cm.sup.3 aliquot of the sample to be tested was added to 9.5
cm.sup.3 of the phosphoric acid buffer. Copper was deposited onto
the working electrode at a potential of -1.5 V vs. SCE, for 15 s
with stirring. The potential was then swept -1.0 V to +0.6 V vs.
SCE with a copper stripping peak observed at ca. -0.05 V vs. SCE.
Standard additions of 1.0 .mu.M Cu(II) were then added over the
range 1.0-10.0 .mu.M and a corresponding addition plot was
constructed and used to calculate the background Cu(II)
concentration in the original sample.
Removal of Copper from River Water
[0089] A 10 cm.sup.3 sample of River Cherwell water (untreated) was
analysed using the SWV copper stripping protocol outlined above and
found to have a Cu(II) concentration of ca. 30 .mu.M which is just
above the EPA limit fo 1.3 mg L.sup.-1 or 20.1 .mu.M and was
therefore used without spiking the Cu(II) concentration. Again the
sample was exposed to 10 mg of CysMeO-GC and analysed at various
intervals for one hour to measure the remaining Cu(II)
concentration. FIG. 7 shows the resulting removal of Cu(II) from
the sample.
EXAMPLE 7
Removal of Arsenic from Water Using PCcarbon and CysMeO-GC
Powder
Reagents and Chemicals
[0090] All chemicals used were of analytical grade and were used as
received without any further purification. These were: sodium
(meta) arsenite (Fluka, +99.0%) and nitric acid (Aldrich, 70%,
double distilled PPB/Teflon grade with trace metal impurities in
parts per trillion determined by ICP-MS). All solutions were
prepared with deionised water of resistivity not less than 18.2
M.OMEGA. cm (Vivendi water systems). A sample of drinking water was
obtained from Bangladesh.
Instrumentation
[0091] Voltammetric measurements were carried out using a
.mu.-Autolab III (ECO-Chemie) potentiostat. All measurements were
conducted using a three electrode cell. The working electrode was a
gold micro disk electrode (1 mm diameter), which was constructed in
house by sealing a gold wire into Teflon housing. The counter
electrode was a bright platinum wire, with a saturated calomel
electrode (Radiometer) as the reference. The gold electrode was
polished using a 0.1 .mu.m alumina slurry on a soft lapping
pad.
[0092] An ultrasonic horn, model CV 26 (Sonics and Materials Inc.)
operating at a frequency of 20 kHz fitted with a 3 mm diameter
titanium alloy microtip (Jencons) was used for sonovoltammetric
studies. The intensity of the ultrasound was determined
calorimetrically (Banks et al, Phys. Chem. Chem. Phys. 2004, 6,
3147; Magulis et al, Russ. J Phys. Chem. 1969, 43, 592; and Magulis
et al, Ultrasonic. Sonochem., 2003, 10, 343) and was found to be 57
Wcm.sup.-2 at 10%. The working electrode was placed in a face-on
arrangement to the ultrasonic horn and the horn was immersed beyond
the shoulder of the stepped tip to ensure that ultrasound was
efficiently applied to the solution. For arsenic detection the
voltammetric curves were baseline corrected using autolab software,
which utilises a third-order polynomial correction.
Removal of Arsenic Using PCcarbon
[0093] Polycysteine-derivatised carbon powder was tested for its
ability to complex As(III) in pure water. As(III) concentrations
were determined using anodic stripping voltammetry (ASV) at a gold
electrode assisted by ultrasound during the deposition process.
Power ultrasound to significantly enhance the sensitivity of
arsenic detection using ASV at a gold electrode. The optimised
conditions reported in Simm et al, Electroanalysis 2005, 17, 335
were used. A control experiment was performed before each sample
was exposed to the complexing ligands to ensure the concentration
of As(III) determined by the standard additions method was correct
to within the detection limits of the procedure.
[0094] A 1.1 mM solution of As(III) was prepared from sodium (meta)
arsenite dissolved in ultra pure water at pH 5.4, 25 mL of the
solution was placed in a stirred flask to which 10 mg of the
polycysteine carbon powder (PCcarbon) was added. At intervals of
10,30 and 60 minutes, a 50 .mu.L sample was taken from the
solution, which was then diluted down into 0.1 M nitric acid to
trace levels for analysis. The analysis was performed by holding
the gold electrode at -0.6 V (vs. SCE) for 60 s, ultrasound was
used during this period at a horn to tip distance of 20 mm and an
amplitude of 5%. The potential was then swept positively to 1 V
(vs. SCE) from the deposition potential at a scan rate of 100 mV/s,
revealing an arsenic stripping signal at .about.0.1 V (vs. SCE).
For each analysis this initial value was measured 3 times and an
average value calculated. Additions of 2.4.times.10.sup.-7 M
As(III) were then performed each measurement which was repeated
three times in order to determine the original concentration of
As(III) present by the standard addition method.
[0095] FIG. 8 shows the reduction in As(III) concentration over
time, after 60 minutes of stirring the concentration of As(III) has
dropped from 1.1 mM to 0.7 mM a 36% decrease, a first order
exponential decay line has been fitted through the points. The
solution was then left for a period of 20 days without further
stirring after this time the concentration was found to have
dropped to 0.55 mM.
Removal of Arsenic Using CysMeO-GC powder
[0096] A 0.98 mM solution of As (III) was prepared from sodium
(meta) arsenite dissolved in ultra pure water at pH 5.4, 25 mL of
the solution was placed in a stirred flask to which 10 mg of the
Cys-GC powder was added. At intervals of 10, 20 and 60 minutes, a
50 .mu.L sample was taken from the solution which was then diluted
down in 0.1 M nitric acid to trace levels for analysis.
[0097] FIG. 9 shows the reduction in As(III) concentration over
time, after 60 minutes of stirring the concentration of As(III) has
dropped from 0.98 mM to 0.7 mM a 28.6% decrease. The solution was
then left 3 days without further stirring however no further
decrease in arsenic concentration was found after this time.
[0098] Experiments were then carried out at trace levels such that
would be expected to be found in drinking water from areas such as
Bangladesh (Anawar et al, Environment International 2002, 27, 597).
A sample was prepared to an As(III) level of 200 ppb (2.66 .mu.M) 4
times greater than the Bangladeshi limit of 50 ppb. 200 mg of
CysMeO-GC powder was then placed in 25 mL of the sample which was
then stirred for a specified length of time before filtration of
the CysMeO-GC powder using filter paper in order to stop the
complexation of As(III) by cystiene. The sample was then diluted
1:1 into a 0.1 M nitric acid solution for analysis.
[0099] FIG. 10 shows that after only ten minutes the arsenic
concentration has been significantly reduced from 200 ppb to 77
ppb, and after 30 minutes the level has dropped to 55 ppb. Analysis
at 60 minutes shows that the concentration of arsenic has remained
constant at this level (a 73% decrease) leaving the concentration
of As(III) present just above the Bangladeshi safe drinking
limit.
[0100] A real sample was then used to test the ability of the
CysMeO-GC powder to complex arsenic in an authentic Bangladeshi
well water sample. The sample was first tested by the ASV method to
determine the concentration of As(III) present. However, the
concentration of As(III) was found to be below the detectable limit
(1.times.10.sup.-8 M), and so the water sample was spiked to a
value of 120 ppb for use in the experiments. As in the experiments
described above, 200 mg of the CysMeO-GC powder was added to 25 mL
of the water sample which was then stirred for a specified time (5,
10, 30 and 45 minutes), before being filtrated to remove the powder
from the solutions. Once again the sample was diluted 1:1 into 0.1
M nitric acid for the analysis experiments.
[0101] FIG. 11 shows the results of the analysis fitted to a first
order exponential decay. After only 5 minutes of stirring the
concentration of arsenic present had dropped by 47% to 64 ppb, at
10 minutes the concentration is found to have dropped further by
69% to 38 ppb (i.e. 12 ppb below the Bangladeshi safe drinking
limit). After 45 minutes, the drop in concentration has levelled
off at 34 ppb, or 28% of the original value. As the analysis was
conducted in a real sample rather than pure water the experiment
was exposed to many trace metals generally found in Bangladeshi
water supplies (copper, lead, mercury etc; Anawar et al,
Environment International 2002, 27, 597). FIG. 12 shows the ASV
plots from the analysis of the 30 minute sample, a large stripping
wave can be seen at approximately 0.4 V vs. SCE, due to one of
these contaminants.
EXAMPLE 8
Derivatisation of Carbon Powder with L-cysteine Methyl Ester
Reagents and Equipment
[0102] All reagents were purchased from Aldrich, with the exception
of the glassy carbon microspherical powder (Alfa Aesar, Type I,
diameter 10-20 .mu.m) and potassium chloride (Reidel de Haen) and
were of the highest commercially available grade and used without
further purification. All aqueous solutions were prepared using
deionised water with a resistivity not less than 18.2 M.OMEGA. cm
(Vivendi Water Systems). pH measurements were performed using a
Hanna Instruments pH213 pH meter.
[0103] X-ray photoelectron spectroscopy (XPS) was performed using a
VG clam 4 MCD analyser system, using X-ray radiation from the Al
K.alpha. band (hv=1486.7 eV). All XPS experiments were recorded
using an analyser energy of 100 eV with a take-off angle of
90.degree.. The base pressure in the analysis chamber was
maintained at no more than 2.0.times.10.sup.-9 mbar. Each carbon
powder sample was mounted on a stub using double-sided adhesive
tape and then placed in the ultra-high vacuum analysis chamber of
the spectrometer. To prevent samples becoming positively charged
when irradiated due to emission of photoelectrons, the sample
surface was bombarded with an electron beam (10 eV) from a "flood
gun" within the analysis chamber of the spectrometer. Note that the
peak positions reported have not been corrected relative to the C 1
s literature value of 286.6 eV to account for the effect of the
flood gun on the peak positions of spectral lines. Analysis of the
resulting spectra was performed using MicroCal Origin 6.0.
Assignment of the spectral peaks was made using the UKSAF and NIST
databases.
[0104] Combustion analysis on samples of CysOMe-carbon was carried
out by determining the percentage elemental content of C, N and S
using standard techniques and equipment.
Coupling of L-cysteine Methyl Ester to Carbon Powder
[0105] Carboxyl moieties were introduced onto the graphite surface
by oxidising oxygen-containing surface groups (e.g. hydroxyl and
quinonyl moieties), which are known to decorate edge-plane defect
sites on graphite surfaces, by stirring graphite powder in
concentrated nitric acid (HNO.sub.3) for 18 hours. The oxidised
graphite powder was then washed with copious quantities of pure
water until the washings ran neutral in order to remove any nitric
acid from the powder sample.
[0106] Modification of graphite powder was then achieved as
follows. 2 g of oxidised graphite powder was stirred in 10 cm.sup.3
of thionyl chloride (SOCl.sub.2) for 90 minutes in order to convert
the surface carboxyl groups to the corresponding acyl chloride
moieties, after which time the resulting material was washed with
dry chloroform to remove any unreacted thionyl chloride impurities.
Next, the powder was suspended in 10 cm.sup.3 of dry chloroform
containing 0.5 g of cysteine methyl ester hydrochloride. 0.27
cm.sup.3 of dry triethylamine was added to this suspension drop
wise and the reaction mixture stirred at room temperature for 12
hours under an inert argon atmosphere. Finally, the resulting
modified graphite powder ("CysOMe-carbon") was washed with copious
quantities of chloroform, acetonitrile acetone and pure water in
order to remove any unreacted species.
Characterisation of CysOMe-carbon Powder
[0107] XPS was used to determine how much CysOMe had been
covalently attached to the graphite surface. A sample of the
CysOMe-carbon powder was mounted in the XPS spectrometer and a scan
was performed from 0-1200 eV as shown in FIG. 13. Peak assignments
were carried out using the UKSAF and NIST databases.
[0108] The percentage surface elemental composition was calculated
from the areas under each peak in the wide spectrum adjusted by
each elements individual X-ray cross-sectional area. Taking into
account the relevant atomic sensitivity factors for the various
elements it was found that the CysOMe comprises ca. 10% of the
surface elements with a variation between different sample
preparations of .+-.3%. This surface coverage is in good agreement
with that obtained using combustion analysis which gave a surface
coverage of CysOMe as being 10-14% and is approximately twice that
for CysOMe-GC powder.
[0109] XPS analysis was also performed on samples of the
CysOMe-carbon powder after exposure to either Cu.sup.II, Cd.sup.II
or As.sup.III solutions for sufficient times for the uptake of
metal ions to be complete (see sections below). FIG. 14 shows the
resulting XPS spectrum of CysOMe-carbon after exposure to
As.sup.III over the region where the As 3s and 3P.sub.3/2 and the S
2s and 2P.sub.3/2 spectral peaks are observed. The ratio of
As.sup.III to CysOMe (as measured by the sulfur spectral line
areas) were found to be approximately 1:1 after taking the relative
atomic sensitivity factors into account. The XPS results for the
other metals studied show a similar stoichometric relationship.
EXAMPLE 9
Detection and Removal of Various Metal Ions Using CysOMe-carbon
Powder Reagents and Equipment
[0110] Electrochemical measurements were performed using a
.mu.-Autolab computer controlled potentiostat (EcoChemie). A three
electrode cell with a solution volume of 10 cm.sup.3 was used
throughout. The working electrode consisted of either a glassy
carbon (GC, 3 mm diameter, BAS), a square boron doped diamond
electrode (BDD, 3 mm.times.3 mm, Windsor Scientific Ltd) or gold (1
mm diameter, GoodFellow) macrodisc electrode. A bright platinum
wire (99.99% GoodFellow) acted as the counter electrode and either
a silver wire pseudo-reference electrode (99.99% GoodFellow) or a
saturated calomel electrode reference electrode (SCE, Radiometer)
completed the three-electrode assembly. All solutions were degassed
using pure N.sub.2 (BOC Gases) for 20 minutes prior to any
electrochemical experiment being performed.
[0111] Inductively coupled plasma atomic emission spectroscopic
(ICPAES) determination of As.sup.III concentration in solution was
analysed with the Perkin Elmer Optima 5300DV emission ICP
instrument. The recommended emission wavelength was 188.979 nm and
axial view is recommended for the best detection. As this is below
the 200 nm threshold the optics were purged at a high flow of argon
to minimise any absorption of light by water and air.
[0112] The As.sup.III calibration, using 5 points (0, 50, 100, 150,
200 ppb), gave a correlation coefficient 0.9993, and the limit of
detection, defined as 3 times the standard deviation of the blank,
averaged from 4 blank checks each measured in 3 replicates, was
found to be 9.78 ppb or 0.0098 ppm. The Perkin Elmer expected value
is 1 to 10 ppb for this wavelength so the sensitivity is
acceptable. The blank check solutions gave between 2.0 and 4.5 ppb
for 4 checks.
Thermodynamics and Kinetics of Cu.sup.II and Cd.sup.II Removal
Using CysOMe-carbon Powder
[0113] The efficacy of CysOMe-carbon powder to the removal of the
heavy metal ions Cu.sup.II, Cd.sup.II and As.sup.III was
determined. Concentration-time profiles were constructed for the
removal of either Cu.sup.II from pH 2.0 solution or Cd.sup.II from
pH 5.0 solution by stirring 25 mg of the modified carbon powder in
25 cm.sup.3 of solutions of varying concentration for varying
amounts of time. The concentration ranges used varied between 5
.mu.M and 500 .mu.M, with the exact solution being determined using
the LSASV analysis prior to commencing the experiments with
graphite powder, and the stirring times were between 2 and 30
minutes in duration.
[0114] Comparison of the concentration-time profiles for the uptake
of either Cu.sup.II or Cd.sup.II between CysOMe-carbon and
CysOMe-GC demonstrates that, in each case, the modified carbon
powder removed a greater amount of the metal ions in a more rapid
fashion, as shown in FIG. 15. This can be attributed to the greater
surface coverage of graphite powder with CysOMe than glassy
carbon.
[0115] The experimental data were analysed using both the Langmuir
and the Freundlich isotherm models. A comparison of the
thermodynamic parameters, K' and n, obtained for both CysOMe-carbon
and CysOMe-GC for Cu.sup.II and Cd.sup.II uptake is given in Table
2. K' and n are Freundlich constants relating to the maximum
adsorption capacity; the larger the value of K' and the smaller the
value of n, the higher the affinity of the adsorbent towards the
adsorbens.
TABLE-US-00002 TABLE 2 Modified carbon powder Metal ion K'/L
g.sup.-1 n CysOMe-GC Cu.sup.II 0.182 1.25 CysOMe-carbon Cu.sup.II
0.136 0.809 CysOMe-GC Cd.sup.II 0.098 0.90 CysOMe-carbon Cd.sup.II
0.167 1.18
[0116] The rate of metal ion adsorption by the CysOMe-carbon was
determined at each concentration studied using the initial rate of
metal ion adsorption from the corresponding concentration-time
profile. The average adsorption rate constant, k.sub.ads, of both
Cu.sup.II and Cd.sup.II by both CysOMe-GC and CysOMe-carbon is
shown in Table 3 for comparison.
TABLE-US-00003 TABLE 3 Modified carbon powder Metal ion
k.sub.ads/cm s.sup.-1 CysOMe-GC Cu.sup.II 2 .times. 10.sup.-4
CysOMe-carbon Cu.sup.II 6 .times. 10.sup.-4 CysOMe-GC Cd.sup.II 3
.times. 10.sup.-4 CysOMe-carbon Cd.sup.II 6 .times. 10.sup.-4
[0117] The faster adsorption kinetics of CysOMe-carbon powder
compared to the CysOMe-GC powder reflect the increased surface
coverage of CysOMe on the graphite particles, which is
approximately twice that of the GC microspheres.
Adsorption of As.sup.III Ions by CysOMe-carbon Powder
[0118] The uptake of As.sup.III ions by CysOMe-carbon powder was
measured as follows. 40 mg of the modified carbon powder was
stirred in 20 cm.sup.3 solution containing varying concentrations
(10 to 150 .mu.M) of arsenic for varying times ranging from a few
minutes to several hours. The powder was then filtered off and the
solution analysed using LSASV to determine the concentration of
As.sup.III remaining. A set of samples that had been analysed by
the LSASV method were then analysed for their As.sup.III
concentration using ICP-AES. The results of the ICP-AES analysis
were found to be in good agreement (within 5%) with those obtained
by LSASV, demonstrating that the electroanalytical protocol
produced accurate and reliable results.
Removal of Trace Amounts of As.sup.III Using CysMeO-carbon
Powder
[0119] 40 mg of CysOMe-carbon powder was stirred in 20 cm.sup.3 of
a solution whose initial As.sup.III concentration was determined to
be ca. 70 ppb for varying times up to 30 minutes, and the
concentration of As.sup.III remaining in the solution monitored
using the trace analysis protocol as described above.
[0120] FIG. 16 shows the resulting concentration time profile. The
initial concentration of As.sup.III was reduced to below the WHO
limit of 10 ppb within 10 minutes of exposure to the small amount
of CysOMe-carbon, and was reduced below the limit of detection of
this methodology after 20 minutes of exposure.
Determination of Cd.sup.III Uptake by CysMeO-carbon Powder
[0121] The concentration of Cd.sup.II remaining in a sample after
exposure to CysOMe-carbon powder was determined using a LSASV
protocol at a boron doped diamond electrode (BDD) developed by
Banks et al (Talanta 2004, 62, 279) in pH 5.0 sodium acetate
buffer. LSASV analysis was carried out using the following
parameters: the BDD electrode was held at a deposition potential of
-1.5 V vs. SCE for 60 seconds with stirring. The potential was then
swept from -1.2 V to -0.1 V vs. SCE at a scan rate of 0.1
Vs.sup.-1. A cadmium stripping peak was observed at ca. -0.8 V vs.
SCE.
[0122] Prior to analysing samples with unknown concentrations of
Cd.sup.II the linear range was determined using the standard
additions method to a sample consisting of blank acetate buffer.
The results show that the LSASV analytical protocol produced a
linear detection range from 1 to 20 .mu.M with a limit of detection
(based on 3.sigma.) of 0.96 .mu.M. Where necessary, samples were
diluted prior to analysis so that their Cd.sup.II concentration
fell within this linear range.
[0123] Standard 1 .mu.M Cd.sup.II additions were then added to the
sample being analysed and the unknown Cd.sup.II concentration was
determined by constructing a standard addition plot, as shown in
FIG. 17. The analysis was repeated three times and the Cd.sup.II
concentration remaining in the sample was calculated as the average
of the three results.
Determination of Cu.sup.II Uptake by CysMeO-carbon Powder
[0124] The Cu.sup.II concentration in a sample was determined using
the standard addition method described above and an LSASV protocol
using the following protocol. Cu.sup.II analysis was performed in
0.1 M H.sub.3PO.sub.4, pH 2.0, using a GC working electrode and a
Ag pseudo-reference electrode to avoid the formation of copper(I)
chloride precipitates during the electrodeposition (which could
otherwise form if a SCE reference electrode was used and are
problematic for the LSASV analysis). A copper stripping peak could
be observed at ca. -0.1 V vs. Ag. The linear analytical
concentration range, using standard additions of 1 .mu.M Cu.sup.II,
was found to be 2 to 20 .mu.M; therefore all samples were diluted
to fall within this range where necessary. LSASV was performed
using a deposition potential of -1.5 V vs. Ag, deposition time 30
s, scan rate 100 mVs.sup.-1 and scanning from -1.5 V to +0.8 V vs.
Ag.
Determination of As.sup.III Uptake by CysMeO-carbon Powder
[0125] LSASV was performed in a solution, 10 cm.sup.3 in volume, of
0.1M HCl (pH 1.0) using a gold working electrode (diameter 1 mm)
with a SCE acting as the reference electrode. The LSASV analysis
was carried out on samples of relatively high concentration using
the following parameters: deposition potential -0.3 V vs. SCE,
deposition time 60 s with stirring for the first 5 s. Then, LSASV
voltammetry was performed from -0.3 V to +0.4 V vs. SCE at 100
mVs.sup.-1, step potential 5 mV. Standard 2.2 .mu.M additions (5
.mu.L of a 4.4 mM standard solution) were then added, and the
unknown sample concentration determined form a standard addition
plot. The linear range for As.sup.III detection was found to be 2
to 20 .mu.M with a limit of detection (based on the 3.sigma. value)
of 1.25 .mu.M. Where necessary, solutions were diluted so that
their concentration fell within this range prior to analysis.
[0126] For the trace analysis work, the protocol was modified
slightly. The solution was stirred throughout the entire 60 s
deposition time with all other parameters identical to those
described above. The standard As.sup.III solution was diluted so
that a 5 .mu.L aliquot added to the analysis sample corresponded to
a 0.22 .mu.M standard addition and the resulting voltammetry is
shown in FIG. 18. The linear range was determined to be 0 to 2.2
.mu.M with a limit of detection of 0.03 .mu.M therefore it was not
necessary to dilute the samples prior to analysis.
* * * * *